Interleukin-19 impairment in active Crohn's disease patients

Abstract

The exact function of interleukin-19 (IL-19) on immune response is poorly understood. In mice, IL-19 up-regulates TNFα and IL-6 expression and its deficiency increases susceptibility to DSS-induced colitis. In humans, IL-19 favors a Th2 response and is elevated in several diseases. We here investigate the expression and effects of IL-19 on cells from active Crohn's disease (CD) patient. Twenty-three active CD patients and 20 healthy controls (HC) were included. mRNA and protein IL-19 levels were analyzed in monocytes. IL-19 effects were determined in vitro on the T cell phenotype and in the production of cytokines by immune cells. We observed that unstimulated and TLR-activated monocytes expressed significantly lower IL-19 mRNA in active CD patients than in HC (logFC = -1.97 unstimulated; -1.88 with Pam3CSK4; and -1.91 with FSL-1; p<0.001). These results were confirmed at protein level. Exogenous IL-19 had an anti-inflammatory effect on HC but not on CD patients. IL-19 decreased TNFα production in PBMC (850.7 ± 75.29 pg/ml vs 2626.0 ± 350 pg/ml; p<0.01) and increased CTLA4 expression (22.04 ± 1.55% vs 13.98 ± 2.05%; p<0.05) and IL-4 production (32.5 ± 8.9 pg/ml vs 13.5 ± 2.9 pg/ml; p<0.05) in T cells from HC. IL-10 regulated IL-19 production in both active CD patients and HC. We observed that three of the miRNAs that can modulate IL-19 mRNA expression, were up-regulated in monocytes from active CD patients. These results suggested that IL-19 had an anti-inflammatory role in this study. Defects in IL-19 expression and the lack of response to this cytokine could contribute to inflammatory mechanisms in active CD patients.

Figure 1. Heat map image of IL-10 family cytokines.

Monocytes mRNA was extracted from HC (n = 3) and active CD patients (n = 3) after 6 h of culture with medium in the absence or presence of Pam3CSK4 and FSL-1. A) Green denoted down-regulated and red up-regulated genes. B) logFC of IL-19 mRNA expression between CD and HC are shown. **p<0.01; ***p<0.001.

Figure 2. IL-19 production after TLR activation in PBMC cultures from active CD patients (n = 3) and HC (n = 6).

PBMC were cultured 24(A) and 48 h (B) with different TLR ligands (LPS, Pam3CSK4 or FSL-1) and IL-19 production was determined by ELISA as described in Materials and Methods. The viability of PBMC was determined by Trypan Blue (>99%). *p<0.05; **p<0.01; ***p<0.001.

Figure 3. rhIL-19 down-regulates TNFα production in cultures from HC after LPS activation.

PBMC from HC and active CD patients were cultured with LPS (HC n = 10 and CD n = 5), Pam3CSK4 (HC n = 10 and CD n = 5) and FSL-1 (HC n = 5 and CD n = 3) for 24 h with or without rhIL-19 (400 ng/ml). A and B) TNFα and IL-10 production in cultures from HC after TLR activation with or without rhIL-19. D and E) TNFα and IL-10 production in cultures from active CD patients after TLR activation with or without rhIL-19. C and F) TNFα production from HC and active CD patients cultured with rhIL-10 (200 ng/ml). *p<0.05.

Figure 4. rhIL-19 up-regulates IL-4 production in activated T cells.

PBMC from HC (n = 6) and active CD (n = 6) were activated and expanded with anti-CD3+anti-CD2+anti-CD28 as described in Materials and Methods. Supernatants were removed and IL-4 and IFNγ content were analyzed by ELISA. A) A) Up-regulation of IL-4 by rhIL-19 in HC cultures. B) IFNγ content in HC and CD patients in the presence or absence of rhIL-19.*p<0.05.

Figure 5. rhIL-19 up-regulates CTLA4 expression on CD4+CD25+CD127

− cells. PBMC from HC (n = 4) and active CD (n = 3) were activated with anti-CD3+anti-CD2+anti-CD28 as described in Materials and Methods. The expression of CTLA4 (A) and GITR (B) was determined on CD4+CD25+CD127− cells by flow cytometry. *p<0.05.

Figure 6. IL-10 regulates IL-19 expression.

PBMC from A) HC (n = 3) and B) active CD patients (n = 3) were activated 24 h with TLR ligands (LPS, Pam3CSK4 and FSL-1) in the presence of rhIL-10 (200 ng/ml) and blocking anti-IL10 (500 ng/ml). Results are expressed as: (IL19 production with TLR+rhIL-10 or anti-IL10-IL-19 production with TLR)/IL-19 production with TLR.

Figure 7. Three miRNAs targeting IL-19 are up-regulated in active CD patients.

miRNA expression in purified monocytes from HC (n = 3) and active CD patients (n = 5) were determined by RT-PCR as described in Materials and Methods. Different expression is shown as Delta Ct values. **p<0.01; ***p<0.001.